Fusaric acid decreases p53 expression by altering promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells

Fusaric acid (FA) is a food-borne mycotoxin that mediates toxicity with limited information on its epigenetic properties. p53 is a tumour suppressor protein that regulates cell cycle arrest and apoptotic cell death. The expression of p53 is regulated transcriptionally by promoter methylation and post-transcriptionally by N-6-methyladenosine (m6A) RNA methylation. We investigated the effect of FA on p53 expression and its epigenetic regulation via promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells. HepG2 cells were treated with FA [0, 25, 50, 104, and 150 mu g/ml; 24 h] and thereafter, DNA, RNA, and protein was isolated. Promoter methylation and expression of p53 was measured using qPCR and Western blot. RNA immuno-precipitation was used to determine m6A-p53levels. The expression of m6A methyltransferases (METTL3andMETTL14), demethylases (FTOandALKBH5), and readers (YTHDF1-3andYTHDC2) were measured using qPCR. FA inducedp53promoter hypermethylation (p< 0.0001) and decreasedp53expression (p< 0.0001). FA decreased m6A-p53levels (p< 0.0001) by decreasingMETTL3(p< 0.0001) andMETTL14(p< 0.0001); and suppressed expression ofYTHDF1(p< 0.0001),YTHDF3(p< 0.0001), andYTHDC2(p< 0.0001) that ultimately reduced p53 translation (p< 0.0001). Taken together, the data shows that FA epigenetically decreased p53 expression by altering its promoter methylation and m6A RNA methylation in HepG2 cells. This study reveals a mechanism for p53 regulation by FA and provides insight into future therapeutic interventions.